Separation of amino acids, amino-acid-based monomer, and process for the preparation thereof, as well as polymer material and process for the preparation thereof

ABSTRACT

A process for separating a selected amino acid (enantiomer) from a mixture of different compounds including other amino acids is disclosed, in which process the mixture is contacted with a polymer material which is composed of cross-linked, amino-acid-based monomer units, said polymer material containing a molecular print of the selected amino acid. In the molecular print there is also bound a diastereomeric complex between the selected amino acid (enantiomer), a divalent metallic ion and the amino-acid-based monomer unit. Also the amino-acid-based monomer unit and a process for preparation thereof, as well as a polymer material composed of the amino-acid-based monomer unit and a process for the preparation thereof are disclosed.

This is a 371 of PCT SE92/00878, filed Dec. 18, 1992.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for separating amino acids,an amino-acid-based monomer, and a process for the preparation thereof,as well as a polymer material, and a process for the preparationthereof.

2. Description of the Related Art

Amino acids are structural elements of one of the most important typesof molecules in nature, viz. proteins. It is most essential that aminoacids of high purity are available, inter alia, as the starting materialin different syntheses and as components in infusions.

Moreover, most amino acids are chiral, and with few exceptions only oneenantiomer is of interest. The access to efficient purification methodsfor amino acids is essential in order to prepare such compounds of high,especially optical, purity.

Today a number of fundamentally different processes are available forpurifying amino acids with regard to both the type of amino acid and theenantiomeric form. These processes may be divided into three main types:chemical, chromatographic and enzymatic. However, only a few of them aresuitable for nonderivatized amino acids which are mainly of interest inindustrial applications, since derivatization of such relativelyinexpensive fine chemicals is unrealistic from the economic point ofview.

The present invention relates to a new, preferably chromatographic,process of purifying nonderivatized amino acids.

The molecular imprinting technique implies preparing a synthetic polymerof desired affinity for a certain molecule (the so-called printmolecule) (See B. Ekberg and K. Mosbach, Trends in Biotechnology, Vol.7, 92-96, 1989; G. Wulff, American Chemical Society Symp. Ser., Vol.308, 186-230, 1986). The polymer is composed of monomer units andintermediate cross-linking agents. The network contains a print of theprint molecule. The preparation can be divided into three main steps, asshown in FIG. 1. First, there are formed chemical interactions betweenthe monomers (of the same or different chemical structures) and theprint molecule in a selected solvent. These interactions may be bothnoncovalent and covalent. After adding cross-linking agents and aninitiator, there is formed in the second step a polymer round thecomplex of monomer and print molecule. This results in a "mould" roundthe print molecule. The last step comprises extraction for removing theprint molecule from the polymer, whereby an affinity seat remains in thepolymer. This affinity seat is a molecular print of the print moleculein the polymer.

The molecular print means that a form of adsorbtive "memory" for theprint molecule has been formed in the polymer. This implies that theprint molecule will be better adsorbed to the polymer than a moleculewhich is structurally related to the print molecule (see FIG. 2).Depending on the type of print molecule, a substrate- orenantio-selective polymer is obtained. This can then be used forspecific separation of the print molecule in a subsequent separationprocess, e.g. liquid chromatography. In the extraction step, the printmolecule can be recovered.

Fundamentally, the functional monomer and also the cross-linking agentare selected according to the interactions that are desired between thisand the print molecule. The interactions can be either noncovalent or(reversibly) covalent. The covalent bonding is not as general as thenoncovalent. Furthermore, more drastic chemical conditions must beapplied to remove the print molecule during the extraction step. Whennoncovalent bondings exist, significantly milder extraction conditionscan be applied. The noncovalent bonding is the most general type ofbonding, since there are more interactions between the print moleculeand monomers at the same time as a mixture of different monomers can beused. (See L. I. Andersson, "Molecular recognition in syntheticpolymers; A study of the preparation and use of molecularly imprintedpolymers, thesis, Applied Biochemistry, University of Lund, 1990).

The molecular imprinting technique has been applied to prepare polymershaving the above described selectively adsorptive properties for aminoacid derivatives (but not nonderivatised amino acids which are thesubject matter of this invention) and smaller peptides (See B. Ekbergand K. Mosbach; G. Wulff; L. I. Andersson; D. O'Shannessy, B. Ekberg andK. Mosbach, J. of Anal. Biochem., Vol. 470, 391-399, 1989; L. I.Andersson and K. Mosbach, J. of Chrom., Vol. 516, 313-322, 1990),β-blockers (See L. Fischer, R. M uller, B. Ekberg and K. Mosbach, J. Am.Chem. Soc., Vol. 113, 9358-9360, 1991), carbohydrate derivatives andcarbohydrates (See G. Wulff), ketones (See K. J. Shea and T. K.Dougherty, J. Am. Chem., Soc., Vol. 108, 1091-1093, 1986), etc.

SUMMARY OF THE INVENTION

Purification of nonderivatised amino acids by means of polymers preparedaccording to the molecular imprinting technique has not been reported sofar, inter alia owing to solubility problems in organic solvents. Thepresent invention makes it possible to purify nonderivatised amino acidsby means of polymers prepared according to a method in which themolecular imprinting technique is partly utilised.

A process for separating a large number of different amino acids, bothfrom each other and resolution of the respective racemate, has beendeveloped by V. A. Davankov et al. (See V. A. Davankov, Pure & Appl.Chem., Vol. 54, No. 11, 2159-2168, 1982; V. A. Davankov, J. D. Navratiland H. F. Walton, "Ligand Exchange Chromatography," CRC Press Inc., BocaRaton, Fla., 1988). The process is based on the forming of adiastereomeric complex between an optically active solid phase (e.g.containing an amino acid derivative) and an amino acid enantiomer in thepresence of a metallic ion (e.g. copper (II)), see FIG. 3. Thesediastereomeric complexes vary in stability. This difference can be usedto separate amino acids (enantiomers) in a liquid chromatography processin which the amino acid (enantiomer) forming the weakest diastereomericcomplex is eluted first. Davankov et al have proved that by this method,a number of amino acids (enantiomers) can be separated.

The present invention relates to a new technique of separating aminoacids and their enantiomers by means of a polymer prepared according toa new method which is a combination of the fundamentally completelydifferent molecular imprinting technique and the so-calledDavankov-method. In this manner, the two separating properties, themolecular print of the nonderivatised amino acid enantiomer and thestability of the diastereomeric complex, are combined in one and thesame polymer, which results in increased separation power in the newpolymer (see FIG. 4).

The present invention thus relates to a process for separating aselected amino acid (enantiomer) from a mixture of different componentsincluding other amino acids. In this process, the mixture of amino acidis contacted with a polymer material which is composed of cross-linked,amino-acid-based monomer units, said polymer material containing amolecular print of the selected amino acid (enantiomer), in whichmolecular print a diastereomeric complex may be formed between the aminoacid, a divalent metallic ion and the amino-acid-based monomer unit.

The invention also relates to an amino-acid-based monomer which consistsof N-methacrylamidomethyl-L-proline or N-methacrylamidomethyl-D-proline.

The invention also relates to a process for preparingN-methacrylamidomethyl-L-proline or N-methacrylaminomethyl-D-proline, inwhich L-proline or D-proline, formalin and methacrylamide are condensedunder alkaline conditions.

Moreover, the invention relates to a polymer material for use inseparating a selected amino acid (enantiomer) from a mixture. Thispolymer material is composed of a cross-linked, amino-acid-based monomerunit and contains a molecular print of the selected amino acid, saidmolecular print further containing a diastereomeric complex between theamino acid, a divalent metallic ion and the amino-acid-based monomerunit.

Finally, the invention also relates to a process for preparing a polymermaterial, said process comprising a) preparation of a diastereomericmetallic ion complex between an amino-acid-based monomer unit and theselected amino acid (enantiomer), b) polymerization of thediastereomeric complex in the presence of a cross-linking agent, and c)removal of the selected amino acid from the polymerized diastereomericcomplex for forming a molecular print of the selected amino acid in thepolymer material.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is described by means of, inter alia, the accompanyingfigures and the examples below.

FIG. 1 illustrates the principle of preparing a molecular print in apolymer.

FIG. 2 illustrates the use of the polymer for separating two differentcompounds.

FIG. 3 illustrates a diastereomeric complex between an optically activesolid phase and an amino acid enantiomer in the presence of a copper(II) ion (according to V. A. Davankov; V. A. Davankov, J. D. Navratiland H. F. Walton).

FIG. 4 shows a polymer material according to the invention beforeremoval of the amino acid enantiomer used as a print molecule (to theright in the figure).

FIG. 5 shows the reaction formula for preparingN-methacrylamidomethyl-L-proline.

DETAILED DESCRIPTION OF THE INVENTION

According to the reaction in FIG. 5, a polymerizable (unsaturated)derivative of L-proline is prepared, N-methacrylamidomethyl-L-proline.The method is distinguished by the reactants being simple and readilyavailable substances (proline, methacrylic acid amide and formaldehyde),and by the reaction occurring under alkaline conditions. Similarly, thecorresponding D-enantiomer can be prepared.

When carrying out this reaction, it is important that the pH value isadapted so as to be higher than the pK_(a2) value of proline. Secondaryamino acids, e.g. proline, and similar substances (pipecolic acid,hydroxy proline, allohydroxy proline, azetidine carboxylic acid andporretine) are stable with respect to racemisation under basicconditions (See R. Liardon and M. Friedman, J. Agric. Food Chem., Vol.35, 661-667, 1987), which means that the optical configuration of theamine can be maintained during the reaction. The racemisation will,however, be significant if primary amines are used instead.

The complex between the polymerizable derivative of L-proline, adivalent metallic ion and a selected amino acid (enantiomer) is preparedby intermixing the components at a controlled pH value. If theL-enantiomer of the proline derivative is used in combination withD-proline, the resulting metallic ion complex will be more stable thanif the L-enantiomer of the proline derivative is used in combinationwith L-proline (See V. A. Davankov, V. R. Meyer and Maya Rais,Chirality, Vol. 2, 208-210, 1990). This combination of enantiomers inthe complex introduces a selectivity for D-proline in the resultingpolymer. To attain a selectivity for L-proline, the complex between theD-proline derivative, a divalent metallic ion and L-proline can beprepared in a similar manner. The proline derivative can also be usedfor the preparation of polymers with selectivity for an amino acidenantiomer other than proline. This can be carried out by preparing thecomplex between the proline derivative and an enantiomer of an aminoacid other than proline.

The selected amino acid (enantiomer) can be any naturally occurring orsynthetic amino acid.

A number of different metallic ions can serve as the coordinating centrein the diastereomeric complex. As mentioned above, Cu(II) is the mostfrequently described, but corresponding complexes with Mn(II), Fe(II),Co(II), Zn(II), Cd(II) and Ni(II) have also been described (see V. A.Davankov, J. D. Navratil and H. F. Walton). In view of the industrialapplicability of the present invention, the purification of amino acidscarried out with smaller toxic metallic ions other than Cu(II) is ofgreat interest.

The polymer can be prepared by polymerizing the selected diastereomericcomplex in an organic solvent in the presence of a cross-linking agent,e.g. ethylene glycol dimethacrylate, while using an initiator, e.g.azoisobutyronitrile. The selection of the initiator allows both thermaland photolytic (366 nm) initiation (see D. O'Shannessy, B. Ekberg and K.Mosbach). The complex may also be polymerized in water in the presenceof water-soluble cross-linking agents, e.g. N,N'-methylenebisacrylamide,while using an initiator system based on ammonium peroxodisulphate.

Chromatographic separation of amino acids (enantiomers) on the preparedpolymers can be carried out while using distilled water, water-basedsolutions and organic solvents as mobile phases.

EXAMPLE 1 Preparation of N-methacrylaminomethyl-L-proline

At room temperature, 3.00 g (0.026 mole) L-proline (Sigma), 1.04 g(0.026 mole) NaOH, and 2.21 g (0.026 mole) methacrylic acid amide(Merck) are dissolved in 50 ml of water in a 100 ml round-bottomed flaskfitted with a tight plastic cork and a magnetic stirrer. 2.0 ml (0.026mole) formaldehyde (Merck) is added during cooling with ice/water bath(about +4° C.). The reaction solution is stirred for 4 h and is thenallowed to take room temperature. The reaction process can be followedby means of thin-layer chromatography on DC-Alufolien, Kieselgel 60 F₂₅₄(Merck) with methanol p.a. as the eluant. Detection is carried out bycolouring with iodine. L-proline: R_(F) 0.35, methacrylic acid amide:R_(F) 0.83, N-methacrylamidomethyl-L-proline: R_(F) 0.56, formaldehydeis not coloured with iodine.

The pH of the reaction mixture is adjusted to 8.5 with 2M sulphuricacid, and subsequently the reaction solution is evaporated at reducedpressure. The remaining oil is dried in a desiccator under vacuum aboveNaOH and is then dissolved in 40 ml methanol (Merck, max. 0.01% H₂ O).Na₂ SO₄ is precipitated. Before this salt is filtered off, 10 gmolecular sieve (Merck, 3 Å) is added. The remaining solution isevaporated at reduced pressure to a volume of about 30 ml. This reactionsolution is purified in a chromatographic process on a silica gel column(Kieselgel 60, Merck, 4×60 cm) with methanol as the eluant. Aftercombining suitable fractions and evaporating at reduced pressure, 3.00 g(corresponding to a yield of 60% of the theoretical maximum) uncolouredsubstance is obtained. R_(F) 0.56 (methanol). Melting point: 90°-92° C.[α]_(D) ²² -71.2° (c=0.773 in methanol/distilled water, 3/1).

¹ H-NMR (CF₃ COOD): 1 H, 5.1 ppm, s; 1 H, 4.8 ppm, s; 1 H, 4.1 ppm, d, ²J=12.45 Hz, AB-spin system; 1 H, 4.02 ppm, d, 2J=12.16 Hz, AB-spinsystem, 1 H, 3.73 ppm, m; 1 H, 3.1 ppm, m; 1 H, 4 H, 1.5 ppm, m; 3 H,1.1 ppm, s.

13_(C) -NMR: R--COO⁻, 175.9 ppm; RHN--CO--R, 174.4 ppm; (R₂)(R₁)C═CH₂,138.3 ppm; (R₂)(R₁)C═CH₂, 128.1 ppm; NCH(CH₂)COO⁻), 67.6 ppm; N--CH₂--N, 62.2 ppm; NCH₂ CH₂, 56.2 ppm; CHCH₂ CH₂, 30.6 ppm; CH₂ CH₂ CH₂,24.8 ppm.

Preparation of N-methacrylamidomethyl-L-proline - Cu(II) - D-prolineComplex

1.71 g (7.3 mmole) N-methacrylamidomethyl-L-proline, 1.165 g (7.3 mole)CuSO₄ and 0.84 g (7.3 mole) D-proline are dissolved in 20 ml ofdistilled water. The pH is adjusted by means of 1M NaOH to 6.85. Thesolution is evaporated at reduced pressure and is dried overnight in adesiccator. Then the dry residue is dissolved in 30 ml methanol, andafter filtering off inorganic salts, the methanol solution is evaporatedat reduced pressure. 2.95 g (corresponding to a yield of 98% of thetheoretical maximum) blue-coloured substance is obtained. R_(F) 0.51(methanol). Melting point: 206°-208° C. IR (cm⁻¹): 3500-3200, 2960-2850;1680-1640 (strong); 1610-1550 (strong); 1550-1520; 1470-1430; 1420-1300.

Preparation of Polymer

2.95 g (7.2 mmol) N-methacrylamidomethyl-L-proline - Cu(II) - D-prolinecomplex is dissolved together with 7.77 g (39.2 mmole) ethylene glycoldimethacrylate (Merck) in 13 ml methanol in a test tube. The solution ispassed by nitrogen gas and before the test tube is provided with atight-fitting cork, 95 mg (0.58 mmole) azoisobutyronitrile (Merck) isadded. The polymerization is then allowed to proceed at 65° C.overnight.

The formed polymer is crushed by hand in a mortar and is then ground ina mechanical mortar device (Retsch, Haan, Germany) for 5 min. Thematerial is sieved through a 25 μm sieve. Subsequently the material isallowed to sediment in two turns in 95% ethanol, while removing thesupernatant. The sediment is carefully washed with 1/1 95%ethanol/distilled water and 1M NH₃ in order to remove D-proline and isthen dried in a desiccator overnight. Amino acid analysis afterhydrolysis (6M HCl, 24 h, 110° C.) of a sample of the polymer provedthat the polymer contains 52% of the theoretical amount of the prolinederivative.

Chromotographic Evaluation

The obtained polymer material is packed in an HPLC steel column (100×4.6mm) in distilled water at a pressure of 200 bar. The column is placed inan HPLC device (Kontron, Switzerland) and equilibrated in 1M NH₃ at aflow rate of 0.25 ml/min, and detection is carried out by measuring theabsorbency at 260 nm. 400 μg D,L-proline is injected in 20 μl distilledwater. k'_(L) =0.40, k'_(D) =1.37. Separation factor: 3.4. Moreover, thesame amount of the following racemates is injected; D,L-threonine,D,L-phenylalanine, D,L-serine and D,L-valine. These racemates were notresolved. The separation factor for the corresponding resolution ofD,L-proline with a polymer prepared according to the Davankov method was1.54 (See B. Lefebvre, R. Audebert and C. Quivoron, J. of Liq. Chrom.,1, 761-774, 1978).

EXAMPLE 2 Preparation of N-methacrylamidomethyl-L-proline - Cu(II) -L-serine Complex

1.71 g (7.3 mmole) L-methacrylamidomethyl-L-proline, 1.165 g (7.3 mmole)CuSO₄ and 0.767 g (7.3 mmole) L-serine are dissolved in 20 ml ofdistilled water. The pH is adjusted by means of 1M NaOH to 6.8. Thesolution is evaporated at reduced pressure and dried overnight in adesiccator. Subsequently, the dry residue is dissolved in 30 ml ofmethanol, and after filtering off inorganic salts, the methanol solutionis evaporated at reduced pressure. 2.95 g (corresponding to a yield of98% of the theoretical maximum) blue-coloured substance is obtained.R_(F) 0.53 (methanol). Melting point: 172°-174° C. IR (cm⁻¹): 3500-3200,2960-2850; 1680-1640 (strong); 1610-1550 (strong); 1550-1520; 1470-1430;1420-1300.

Preparation of Polymer

6.8 mmole N-methacrylamidomethyl-L-proline - Cu(II) - L-serine complexis dissolved together with 5.24 g (34 mmole) N,N'-methylenebisacrylamidein distilled water to a total volume of 160 ml. 0.125 g (0.54 mmole)(NH₄)₂ SO₄ is added together with 40 μlN,N,N',N'-tetramethyletylenediamine, and the solution is passed bynitrogen gas for a few minutes. After 4 h at room temperature, agel-like polymer is obtained.

The polymer formed is isolated by filtration and is then allowed tosediment in 5 turns in distilled water, while removing the supernatant.The sediment is carefully washed with 1/1 95% ethanol/distilled waterand distilled water to remove L-serine and is then dried in a desiccatorovernight. Amino acid analysis after hydrolysis (6M HCl, 24 h, 110° C.)of a sample of the polymer proved that the polymer contains 64% of thetheoretical amount of the proline derivative.

Chromatographic Evaluation

The polymer (1.4 g) is suspended in 50 ml 0.1M Cu(CH₃ COO)₂, pH 5.3, isthen washed in 5 turns with distilled water and is packed at a flow rateof 0.30 ml/min in distilled water in a low-pressure chromatographycolumn (8×2.5 cm). 22 mg D,L-serine is applied in 0.5 ml distilledwater, and the separation is carried out with the same eluant and at aflow rate of 0.30 ml/min. The eluate is collected in fractions of 1.45ml. The contents of the two enantiomers in the fractions are determinedby HPLC analysis after derivatising with(+)-1-(9-fluorenyl)ethylchloroformate (See S. Einarsson, B. Josefsson,P. M oller and D. Sanchez, Anal. Chem., Vol. 59, 1191-1195, 1987). Theanalysis showed that the fractions 7, 8, 9, 10 and 11 contained 100%D-serine, the fractions 12, 13, 14 and 15 96% D-serine and 4% L-serine;the fractions 18, 19 and 20 95% D-serine and 5% L-serine.

What is claimed is:
 1. A method for preparing a molecular print polymerfor adsorbing an enantiomer of a chiral nonderivatized amino acid,comprising:(A) preparing a diastereomeric complex between a monomer unitcomprising an amino acid moiety, an enantiomer of a chiralnonderivatized amino acid, and a divalent metallic ion capable offorming a complex between said monomer unit and said enantiomer; (B)polymerizing the monomer moieties of said complex in the presence of acrosslinking agent; and (C) removing said amino acid from saidpolymerized complex, thereby obtaining said molecular print polymer. 2.The method as claimed in claim 1, wherein said chiral nonderivatizedamino acid is naturally occurring.
 3. The method as claimed in claim 1,wherein said chiral nonderivatized amino acid is synthetic.
 4. Themethod as claimed in claim 1, wherein said monomer unit comprising anamino acid moiety is selected from the group consisting ofN-methacrylamidomethyl-L-proline and N-methacrylamidomethyl-D-proline.5. The method as claimed in claim 1, wherein said divalent metallic ionis selected from the group consisting of copper (II), manganese (II),iron (II), cobalt (II), zinc (II), cadmium (II), and nickel (II).
 6. Amolecular print polymer produced by the process of claim
 1. 7. Amolecular print polymer comprising:(A) a crosslinked polymerized monomercomprising an amino acid moiety; (B) a divalent metallic ion capable offorming a complex between said amino acid moiety of said monomer and anenantiomer of a chiral nonderivatized amino acid;wherein said polymercomprises affinity seats for said enantiomer, formed when said monomeris polymerized and crosslinked as a complex of said enantiomer and saiddivalent metallic ion.
 8. The molecular print polymer as claimed inclaim 7, wherein said monomer unit comprising an amino acid moiety isselected from the group consisting of N-methacrylamidomethyl-L-prolineand N-methacrylamidomethyl-D-proline.
 9. The molecular print polymer asclaimed in claim 7, wherein said chiral nonderivatized amino acid isnaturally occurring.
 10. The molecular print polymer as claimed in claim7, wherein said chiral nonderivatized amino acid is synthetic.
 11. Themolecular print polymer as claimed in claim 7, wherein said divalentmetallic ion is selected from the group consisting of copper (II),manganese (II), iron (II), cobalt (II), zinc (II), cadmium (II), andnickel (II).
 12. A process for separating an enantiomer of a chiralnonderivatized amino acid from a mixture thereof, comprising:(A)contacting a mixture comprising an enantiomer of a chiral nonderivatizedamino acid with a molecular print polymer comprising:(1) a crosslinkedpolymerized monomer comprising an amino acid moiety; (2) a divalentmetallic ion capable of forming a complex between said amino acid moietyof said monomer and an enantiomer of a chiral nonderivatized amino acid;wherein said polymer comprises affinity seats for said enantiomer,formed when said monomer is polymerized and crosslinked as a complex ofsaid enantiomer and said divalent metallic ion; wherein said contactingoccurs under conditions sufficient for said enantiomer to form a complexwith said divalent metallic ion of said polymer; and (B) removing theenantiomer depleted mixture from contact with the polymer.
 13. Theprocess as claimed in claim 12, wherein said monomer unit comprising anamino acid moiety is selected from the group consisting ofN-methacrylamidomethyl-L-proline and N-methacrylamidomethyl-D-proline.14. The process as claimed in claim 12, wherein said chiralnonderivatized amino acid is naturally occurring.
 15. The process asclaimed in claim 12, wherein said chiral nonderivatized amino acid issynthetic.
 16. The process as claimed in claim 12, wherein said divalentmetallic ion is selected from the group consisting of copper (II),manganese (II), iron (II), cobalt (II), zinc (II), cadmium (II), andnickel (II).
 17. The process as claimed in claim 12, wherein saidenantiomer of said chiral amino acid is selected from the groupconsisting of D-proline and L-serine.
 18. An amino acid-based monomerselected from the group consisting of N-methacrylamidomethyl-L-prolineand N-methacrylamidomethyl-D-proline.
 19. A method for preparingN-methacrylamidomethyl-L-proline and N-methacrylamidomethyl-D-proline,comprising condensing L-proline or D-proline, formalin, andmethacrylamide under alkaline conditions.